The human gastric adenocarcinoma cell lines AGS (p53 wild type) and Kato 3 (p53 null type) (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI 1640 medium (WelGENE Inc., Daegu, Korea) supplemented with 10% fetal calf serum, and 0.1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2. Antibodies against procaspases 3, cleaved caspase 3, cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-actin, Nrf2, p53, phospho-JNK, JNK were purchased from from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FLAG and β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). The anti-mouse or anti-rabbit secondary antibodies were purchased Cell Signaling Technology. Shikonin, Sp600125 and Ac-DEVD-CHO were obtained from Sigma-Aldrich.
Anti mouse or anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-mouse or anti-rabbit secondary antibodies are laboratory reagents used to detect the presence of primary antibodies in various experimental techniques, such as Western blotting, immunohistochemistry, and flow cytometry. These secondary antibodies are designed to bind specifically to the constant regions of mouse or rabbit primary antibodies, respectively, allowing for the visualization and amplification of the target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (2 mentions)
Anti vimentin rv202, by BD (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti flag and β actin antibodies, by Merck Group (1 mentions)
Ac devd cho, by Merck Group (1 mentions)
Kato 3, by Korean Cell Line Bank (1 mentions)
Rpmi 1640 medium, by Welgene (1 mentions)
Genebox detection system, by Syngene (1 mentions)
Actin hrp, by Santa Cruz Biotechnology (1 mentions)
Startingblock blocking buffer, by Thermo Fisher Scientific (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Myogenin, by Abcam (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
23 protocols using anti mouse or anti rabbit secondary antibody
1
Gastric Adenocarcinoma Cell Line Study
2
Whole-cell Protein Extraction and Western Blot
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Whole-cell extracts were prepared using NP-40 lysis buffer (20 mM Tris–HCl, pH 8.8, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 12 mM Na-deoxycholate). Cell homogenates were incubated for 30 min on ice followed by a clean-up centrifugation at 20,000 × g for 10 min at 4°C. Protein concentration was determined with Pierce BCA Protein Assay Reagent (Thermo Scientific) using bovine serum albumin as a standard. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose (Bio-Rad) membrane. The membrane was blocked with StartingBlock Blocking Buffer (Thermo Scientific) for 1 h followed by incubation with primary antibody: PARP-1 (1:1,000; Cell Signaling, Cat#9532), PAX7 (1:1,000; Abcam, Cat#ab34360), myogenin (1:1,000; Abcam, ab124800), PCNA (1:1,000; Cell Signaling, Cat#2586), Histone H3 (1:1,000; Cell Signaling, Cat#12648P), ATP synthase (subunit alpha) (1:1000, Life Technologies, Cat#459240/G0531), actin-HRP (1:5,000; Santa Cruz Biotechnology, Cat#sc-1616 HRP); followed by incubation with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling). The membrane was developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and visualized in a GeneBox Detection System (Syngene).
3
Signaling Pathway Analysis of NECA and APCP
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5′-(N-Ethylcarboxamido) adenosine (NECA, E2387) and Adenosine 5′-(α, β-methylene) diphosphate (APCP, M3763) were purchased from Sigma. Cells were seeded into 6-well plates at a concentration of 30 × 104 cells/well. After 24 h, cells were treated with NECA (1 μM) for 24 h or APCP (10 μM) for 1 h, then were harvested and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA).
For the human receptor tyrosine kinase (RTKs) assay, we used the phosphorylation antibody array-AAH-PRTK-1 (RayBiotech Inc.). Protein lysates were incubated with the array membrane and protein signal was visualized using a chemifluorescence detection system (Bio-Rad) according to the manufacturer’s protocol. Relative density of specific protein expression was determined using Quantity One software.
Western blot analysis was performed as previously described [43 (link)]. The antibodies used in the analysis were anti-CD73 (D7F9A), anti-pEGFR (Tyr1068, 1H12), anti-pAKT (Ser473, D9E), anti-AKT, anti-pERK (Thr202/204, D13.12.4E), anti-ERK (137 F5), anti-pFak (Tyr397, D20B1), anti-FAK (D2R2E) and anti-CyclinD1 (92G2, all from Cell Signaling Technology, Danvers, MA, USA), anti-EGFR (A-10, Santa Cruz, CA, USA), anti-β-actin and anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology).
For the human receptor tyrosine kinase (RTKs) assay, we used the phosphorylation antibody array-AAH-PRTK-1 (RayBiotech Inc.). Protein lysates were incubated with the array membrane and protein signal was visualized using a chemifluorescence detection system (Bio-Rad) according to the manufacturer’s protocol. Relative density of specific protein expression was determined using Quantity One software.
Western blot analysis was performed as previously described [43 (link)]. The antibodies used in the analysis were anti-CD73 (D7F9A), anti-pEGFR (Tyr1068, 1H12), anti-pAKT (Ser473, D9E), anti-AKT, anti-pERK (Thr202/204, D13.12.4E), anti-ERK (137 F5), anti-pFak (Tyr397, D20B1), anti-FAK (D2R2E) and anti-CyclinD1 (92G2, all from Cell Signaling Technology, Danvers, MA, USA), anti-EGFR (A-10, Santa Cruz, CA, USA), anti-β-actin and anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology).
4
Immunoblotting of Angiogenesis Markers
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All procedures were performed as we previously reported22 (link). The antibodies used in our current study were anti-NRP1 (A-12) (Santa Cruz Biotechnology, CA, USA), anti-HIF-1α (D1S7W), anti-VE-cadherin (D87F2), anti-MMP2 (D8N9Y) (Cell Signaling Technology, Danvers, MA, USA), and anti-Vimentin (RV202) (BD Biosciences, Oxford, UK). Anti-β-actin and anti-mouse or anti-rabbit secondary antibodies from Cell Signaling Technology were used.
5
Western Blot Analysis of Signaling Proteins
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Western blotting analysis was performed as previously described.13 The antibodies used in the analysis were anti‐NRP1 (A‐12), anti‐phospho (p)EGFR (Tyr1068) (1H12), anti‐EGFR (A‐10) (all from Santa Cruz, Santa Cruz, CA), anti‐pAKT (Ser473) (D9E), anti‐AKT, anti‐pFAK (Tyr397) (D20B1), anti‐FAK (D2R2E), anti‐Cyclin D1 (92G2), anti‐MMP2 (D8N9Y), anti‐MMP9 (603H), anti‐Snail (C15D) (all from Cell Signaling Technology, Danvers, MA), anti‐N‐cadherin, anti‐Vimentin (RV202) (both from BD Biosciences, San Jose, CA), anti‐β‐actin and anti‐mouse or anti‐rabbit secondary antibodies (all from Cell Signaling Technology). According to the protein loading marker, we cut the whole membranes into small pieces and incubated them with corresponding specific primary antibody overnight at 4°C. On day 2, after washing four times with 1 × TBST (Tris‐buffered saline–Tween 20), we continued to incubate the membrane pieces with the appropriate secondary antibodies. Image J was used to quantify the band density of the immunoreactive proteins. After we opening our immunoblot images in image J, we transformed the image type to 8‐bit to be recognized and then subtracted the background. Finally, we set the associated measurements and selected our target band to obtain integrated density value for further analysis in Excel.
6
Western Blot Analysis of p53 and GAPDH
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Cells were harvested by scraping from the culture dish and washed twice with ice-cold phosphate buffered saline (PBS). Total protein was extracted from the cell pellet with RIPA lysis buffer (Upstate, Charlottesville, VA). Samples were incubated on ice for 30 min and then centrifuged at 12,000 g for 20 min at 4 °C. Supernatants were collected and mixed with sodium dodecyl sulfate sample buffer, heated at 95 °C for 10 min and separated by polyacrylamide gel electrophoresis and electrical blotted to polyvinylidenedifluoride membranes (Whatman, Inc., Clifton, NJ). Membrane was incubated for 1 h at room temperature in a blocking solution composed of 5% skimmed milk powder dissolved in TBST (0.05% Tween-20, 10 mM Tris, pH 8.0, and 140 mM NaCl). After washing the membrane three times with TBST, proteins were revealed by mouse and rabbit antibodies against p53, or GAPDH by overnight incubation at 4 °C. After three washes with TBST, membranes were incubated with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.). The blots were then subjected to chemiluminescent detection according to the manufacturer’s instructions.
7
Immunoblotting of Protein Samples
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Immunoblotting was performed according to standard techniques. Briefly, protein was diluted to 4 mg/mL in Laemmli buffer. Forty micrograms of protein was electrophoresed on 4–20% PAGEr Gold Precast Gels (Lonza, Walkersville, MD) at 120 V by SDS‐PAGE and transferred onto nitrocellulose membranes (Bio‐Rad, Hercules, CA) at 100 V for 1 h at 4°C. To ensure equal loading, membranes were stained with Ponceau‐S stain and imaged with FOTO/Analyst PC Image software. The resulting signal was quantified and was similar between groups for all membranes. Membranes were destained in 1% TBST (50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Tween 20) for 20 min, blocked in 5% milk in 1% TBST for 1 h, and then exposed to primary antibodies overnight at 4°C (Table 1 ). Membranes were then washed, exposed to appropriate anti‐mouse or anti‐rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA) for 1 h at room temperature, washed in 1% TBST, and incubated with ECL Western Blotting Substrate for 5 min at room temperature (Thermo Fisher Scientific, Inc.). Blots were imaged on X‐ray film (Phenix Research Products, Candler, NC), and the resultant bands quantified with Carestream software. Signal from each band was normalized to mean signal from the control (thermal neutral) group.
8
Protein Expression Analysis by Western Blot
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Western blot was performed as previously described [17 (link)]. The primary antibodies (ITGAL and β-actin) were purchased from Abcam, United States. The anti-mouse or anti-rabbit secondary antibodies were obtained from Cell Signaling Technology, United States.
9
Western Blot Analysis of Protein Expression
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Transfected cells were washed and homogenized in a protein lysis buffer (RIPA buffer). Protein concentrations were quantified using BCA Protein Assay Kit (Pierce). 10 μg of each sample were denaturized at 95°C for 10 min and further processed using the NuPAGE SDSPAGE Gel System (Invitrogen). Proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL, GE Healthcare), which was treated afterwards with 5% skimmed milk powder in TBS Tween 0.1%. Primary antibodies were applied to the membrane according to manufactures’ protocol: anti-p-AKT Ser473 (Cell Signaling), anti-AKT (Cell Signaling), anti-BCL-X S/L (Santa Cruz Biotechnology), anti-cFLIP S/L (Adipogen) anti-COPB2 (novus Biologicals), anti-p-ERK1/2 Thr202/Tyr204 (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-GAPDH (Cell Signaling), anti-KRAS (Santa Cruz Biotechnology), anti-LC3 (Cell Signaling), anti-pSTAT3 (Tyr705) (Cell Signaling), anti-STAT3 (Cell Signaling), anti-PARP (Cell Signaling), anti-XIAP (BD Biosciences). The membrane was washed and incubated with the appropriate horseradish peroxidase-conjugated antiMouse or anti-Rabbit secondary antibodies (Cell Signaling). The chemiluminiscent reaction was initialized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and detected by G:Box Chemi XT4 (Syngene) (Figures 4 , 5 , 6 ).
10
Protein Expression Quantification Protocol
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Total protein extracts were prepared as described previously [19 (link)]. Ob-R (B-3) (sc-8391), GR (41) (sc-136209), GILZ (sc-33780) (Santa Cruz Biotechnology, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich, USA) primary antibodies were used. Western blots were visualized with an anti-mouse or anti-rabbit secondary antibody (Cell Signaling, USA) diluted 1/1000 and enhanced chemiluminescence reagents (GE Healthcare, UK). Western blot band quantification was performed with Image Studio Lite software (USA), and protein expression levels were normalized to the GAPDH level.
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